Concerted inhibition of the transcriptional activation functions of the enhancer‐binding protein NIFA by the anti‐activator NIFL

Abstract

SummaryThe Azotobacter vinelandii NIFL regulatory flavoprotein responds to the redox, energy and nitrogen status of the cell to inhibit transcriptional activation by the s N -dependent enhancer binding protein, NIFA, via the formation of a NIFL±NIFA protein complex. The NIFA protein contains three domains: an Nterminal domain of unknown function; a central catalytic domain required to couple nucleotide hydrolysis to activation of the s N -RNA polymerase holoenzyme; and a C-terminal DNA-binding domain. We report that truncated NIFA proteins that either lack the amino-terminal domain or contain only the isolated central domain remain responsive to inhibition by NIFL but, in contrast to native NIFA, continue to hydrolyse nucleotides when NIFL is present. We also report that NIFL is competent to inhibit the DNAbinding function of NIFA. Taken together, these results suggest that NIFL inhibits NIFA via a concerted mechanism in which DNA binding, catalytic activity and, potentially, interaction with the polymerase are controlled by NIFL in order to prevent transcriptional activation under detrimental environmental conditions.

The Amino-terminal GAF Domain of Azotobacter vinelandii NifA Binds 2-Oxoglutarate to Resist Inhibition by NifL under Nitrogen-limiting Conditions

“…Accordingly, we examined whether a truncated form of NifA lacking the GAF domain, NifA-(191-522), is responsive to 2-oxoglutarate in the presence of NifL. This truncated protein retains the ability to activate transcription and is responsive to NifL (8). For these assays, we measured the formation of open promoter complexes by NifA in the presence of 54 -RNA polymerase holoenzyme and integration host factor at the nifH promoter (5).…”

“…Plasmids for Overexpression-Plasmids pTJ40, pTJ54, pDB737, and pAS1, used for overexpression of NifL His6 , NifL-(147-519) His6 , NifA, and NifA-(191-522), respectively, have been described previously (7,8). Plasmid pTM17 expressing the GAF domain of A. vinelandii NifA with a carboxyl-terminal hexahistidine tag (Av NifA-(1-203) His6 ) was prepared using primers "nifa-3" (5Ј-ATGTTCTCGAAGCCGTACTCGAGG-CGCACCTCGCG-3Ј) and "05-97" (5Ј-GGGAATGCATATGAATGCAAC-CATCC-3Ј) to amplify a fragment containing residues 1-203 with plasmid pDB737 as a template; this resulted in the introduction of a new XhoI site downstream of the coding sequence for residue 203.…”

“…Cultures were grown aerobically in Luria-Bertani medium, and expression from the T7 promoter was induced by addition of isopropyl-␤-D-thioglactopyranoside to 1 mM. A. vinelandii NifA, NifA-(191-522), Av GlnK, K. pneumoniae 54 , and IHF were purified as described previously (8,16). NifL-(147-519) His6 , Av NifA-(1-203) His6 , Av NifA His6 -(1-180), and Kp NifA His6 -(1-178) were purified by metal chelate affinity chromatography.…”

“…Lanes 4,7,10,13,16,19, and 22 contained NifA, ATP, and 2-oxoglutarate incubated for t ϭ 0, 2, 6, 10, 30, 60, and 100 min, respectively. Lanes 5,8,11,14,17,20, and 23 contained NifA, ATP, and 3-oxoglutarate incubated for t ϭ 0, 2, 6, 10, 30, 60, and 100 min, respectively. Lane 2 shows purified NifA as a marker.…”

“…Although nucleotide hydrolysis or phosphotransfer has not been observed, binding of adenosine nucleotides to the GHKL domain plays a key role in transducing environmental signals by enhancing the inhibitory activity of NifL and stabilizing protein-protein interactions between NifL and NifA (5-7). NifA activity is inhibited by NifL via a concerted mechanism in which catalytic activity, DNA binding, and potentially interaction with RNA polymerase are controlled (8).…”